1. Field of the Invention
The present invention relates to site-specific .sup.13 C-enriched reagents for diagnostic medicine for magnetic resonance imaging. The site-specific .sup.13 C-enriched reagents may be represented by the formula: T-L-R. T is a site-specific targeting group which selectively binds to a disease-related target in an animal or human, R is an inert polymer containing repeating .sup.13 C reporting groups which provide a magnetic resonance imaging signal, and L is a linker group which connects the site-specific targeting group to the inert polymer. The site-specific .sup.13 C-enriched reagents are targeted to and capable of identifying, quantifying, and localizing disease-specific loci, such as blood clots, .beta.-amyloid plaques of Alzheimer's disease, and tumors through the use of magnetic resonance imaging. The present invention also pertains to a method for employing the site-specific .sup.13 C-enriched reagents in a living mammal.
2. Description of the Background
Many diagnostic and therapeutic medical procedures for visualizing internal organs for the early detection and treatment of many diseases require the administration of contrast enhancing agents to improve the quality of the procedure. Contrast-enhancing agents are used in Magnetic Resonance Imaging (MRI), Computerized Tomography (CT), and X-ray procedures. Computerized Tomography provides a more sophisticated visualization of tissues and organs than does conventional X-ray techniques. Magnetic Resonance Imaging provides a superior soft tissue differentiation than does Computerized Tomography. Magnetic Resonance Imaging procedures generally employ the nuclear magnetic resonance of hydrogen (.sup.1 H) or fluorine (.sup.19 F). The nuclear magnetic resonance sensitivity of .sup.19 F is nearly equivalent to that of .sup.1 H but the biological background of .sup.19 F is negligible. The usefulness of a contrast enhancing agent depends upon the ease of the synthesis of the agent, the site-specificity of the agent, the resistance to in vivo hydrolysis of the agent, and a sufficient amount of signal from the agent along with a high signal-to-noise ratio.
In radioscintigraphy, a radioactive monoclonal antibody is typically injected into a patient for identifying and localizing a tumor, (reviewed in Bischof Delaloye, A. and Delaloye, B.: Tumor imaging with monoclonal antibodies. Seminars in Nuclear Medicine 25(2):144-164, 1995).
In radioimaging with monoclonal antibodies, a chemically modified (chelate) form of a monoclonal antibody is typically prepared and stored as a relatively stable product. To be used clinically, however, the monoclonal antibody sample must be mixed with a radioactive metal, such as .sup.99m Tc, then purified to remove excess, unbound radioactive metal, and then administered to a patient within 6 hours, (Eckelman, W. C., Paik, C. H., and Steigman, J.: Three approaches to radiolabeling antibodies with .sup.99m Tc. Nuc. Med. Biol. 16: 171-176, 1989). The entire process is cumbersome and dangerous due to the many manipulations requiring use and disposal of radioactivity. There is some health risk and fear accompanying injection of radioactivity into the patient.
Another example of imaging technology is the diagnosis of Alzheimer's disease, which afflicts over 3 million Americans and is increasing in incidence as the number of senior citizens increases. Currently, diagnosis is made by ruling out other causes for the symptoms of memory loss and dementia. The ability to diagnose Alzheimer's disease could also be critical to the development of an effective therapy. In particular, a therapeutic strategy that would arrest or reverse the buildup in the brain of .beta.-amyloid plaques, which has been shown to be the cause of nerve cell loss, would require a means for imaging and measuring these deposits. The chemical, Congo red, has been shown to bind to .beta.-amyloid plaques and a form of Congo red capable of chelating a radioactive metal has been prepared and proposed for use in imaging by radioscintigraphy (Chem. & Eng. News, Jun. 17, 1996, pages 33-34). Alternatively, the protein, tissue plasminogen activator, can be used in a radiolabeled form as a diagnostic reagent to image .beta.-amyloid plaques, U.S. Pat. No. 5,589,154 (Anderson). Still another potential imaging agent is .beta.-amyloid peptide, which can deposit into the plaque. Although .beta.-amyloid plaques are within the brain, they are also present in the small and medium-sized arteries serving the brain, and are uniquely associated with Alzheimer's disease, (Vinters, H. V.; Cerebral amyloid angiopathy. A critical review. Stroke 18:311-324, 1987).
Another example of imaging technology is the diagnosis of blood clots. Despite the frequency of pulmonary thromboembolism and its associated morbidity and mortality, diagnosis remains suboptimal. Similarly, noninvasive detection of both deep vein and cerebral thrombosis is currently difficult. Various radiolabeled proteins, such as antifibrin monoclonal antibodies, (Rosebrough, S. F. and Hashmi, M.: Galactose-modified streptavidin-GC4 antifibrin monoclonal antibody conjugates: application for two-step thrombus/embolus imaging. J. Pharm. Fxp. Ther. 276(2): 770-775, 1996), fibrin-binding domain fragment of fibronectin (Rosenthall. L. and Leclerc, J.: A new thrombus imaging agent. Human recombinant fibrin binding domain labeled with In-ill. Clin. Nucl. Med. 20(5): 398-402, 1995), activated-platelet binding protein fragment (Muto, P., Lastoria, S., Varrella, P., et al.: Detecting deep venous thrombosis with technetium-.sup.99m -labeled synthetic peptide P280. J. Nucl. Med. 36(8): 1384-1391, 1995) and (inactivated) tissue plasminogen activator (De Bruyn, V. H., Bergmann, S. R., Keyt, B. A. and Sobel, B. E.: Visualization of thrombi in pulmonary arteries with radiolabeled, enzymatically inactivated tissue-plasminogen activator. Circulation 92(5): 1320-1325, 1995) have been utilized for imaging thrombi.
The use of non-targeted, stable isotope-enriched contrast reagents, such as .sup.13 C glucose, has been described previously for imaging and for metabolic studies, (Shulman, R. G., Blamire, A. M., Rothman, D. L. and McCarthy, G. Nuclear magnetic imaging and spectroscopy of human brain function. Proc. Natl. Acad. Sci. USA 90(8): 3127-3133, 1993; Sonnewald, U., Gribbstad, I. S., Westergaard, N. Nilsen, G., Unsgard, G., Schousboe, A. and Peterson, S. B. Nuclear magnetic resonance spectroscopy: biochemical evaluation of brain function in vivo and in vitro. Neurotox. 15(3): 579-590, 1994). Also, the feature of targeted binding to a disease-indicating locus has been described for radioactive isotope-enriched reagents (see references above). Furthermore, tumor-localizing reagents containing metals such as gadolinium, which enhance contrast in proton (.sup.1 H) MRI, have been described, (Young, S. W., Qing, F., Harriman, A., Sessler, J. L., Dow, W. C., Mody, T. D., Hemmi, G. W., Hao, Y. and Miller, R. A. Gadolinium(III) texaphyrin: A tumor selective radiation enhancer that is detectable by MRI. Proc. Natl. Acad. Sci. USA 93: 6610-6615; Igarashi, N., Igarashi, S., Fujio, N. and Yoshida, A. Magnetic resonance imaging in the early diagnosis of cavernous sinus thrombosis. Ophthalmologica 209(5): 292-296, 1995; Williams, R. F., Siegle, R. L., Pierce, B. L. and Floyd, L. J. Analogs of synthetic melanin polymers for specific imaging applications. Invest. Radiology 29: S116-119, 1994; Orang-Khadivi, K., Pierce, B. L., Ollom, C. M., Floyd, L. J., Siegle, R. L. and Williams, R. F. New magnetic resonance imaging techniques for the detection of breast cancer. Breast Cancer Res. Treat. 32(1): 119-135, 1994.).
U.S. Pat. No. 4,624,846 (Goldenberg) discloses a method for enhancing the target specificity of antibody localization. The method comprises injecting a second antibody specific to a labeled target-specific antibody to reduce the level of non-targeted circulating specific antibody and thereby increase the localization ratio. Specifically, the method comprises injecting a human subject parenterally with a marker-specific antibody labeled with a pharmacologically inert radioisotope, capable of detection using a photoscanning device, or with a paramagnetic conjugate, capable of detection with a magnetic resonance detector, and subsequently scanning with the device or detector to detect and locate the site of uptake of the labeled antibody by the tumor. The improvement provided by the method comprises injecting the subject parenterally, at a time after injection of the marker-specific antibody sufficient to permit maximum selective uptake by the tumor, and prior to scanning, with a second, non-labeled antibody specific against the marker-specific antibody, in an amount sufficient to decrease the level of circulating labeled marker-specific antibody or fragment by 10-85% within 2-72 hours. Goldenberg discloses the use of numerous antibodies labeled with radionuclides for detection by photoscanning devices and paramagnetic species for detection by a magnetic resonance detector. The method is said to be useful to help determine the location of a tumor which produces or is associated with a cytoplasmic, intracellular, or cell-surface marker substance.
U.S. Pat. No. 5,236,694 (Antich et al. '694) discloses the use of .sup.19 F labelled compounds in methods of NMR imaging and spectroscopy. The compounds comprise a .sup.19 F-containing sensor moiety and a transport polymer, and may also comprise a spacer moiety to separate the sensor moiety and the transport polymer. Specifically, the method comprises administering to a living subject a .sup.19 F labelled NMR agent comprising (a) a transport polymer selected from the group consisting of dextran polymers and aminodextrans, having a molecular weight between approximately 100 d and 500 kd, and antibodies and fragments thereof, and (b) a .sup.19 F-containing sensor moiety selected from the group consisting of fluorinated alkyls, fluorinated acetates, fluoroaniline, and fluoroalkyl phosphonates, in an amount effective to provide a detectable NMR signal. The signal produced by the .sup.19 F labelled NMR agent in the subject is then detected.
U.S. Pat. No. 5,308,604 (Sinn et al.) discloses conjugates composed of a) at least one polyalcohol or a derivatized polyalcohol, b) at least one active agent, c) at least one linker, and d) a protein. The polyalcohols are compounds which are not recognized by the defense system of an organism as exogenous, such as sorbitol or derivatized sorbitol, with at least one OH group being replaced by .sup.19 F, C.sup.19 F.sub.3, mono- or poly-.sup.19 F-substituted C.sub.1 -C.sub.4 alkyl, mono, di-, tri-, tetra- or penta-.sup.19 F-substituted phenyl. The active agent is a compound which is able to emit a signal to an external scanning device and/or is able to have a direct or indirect therapeutic effect on tumor tissue, and preferably is a .sup.19 F, .sup.131 I, or .sup.132 I labeled aromatic compound. The linker is a compound which may be used as a coupling member or spacing member between the protein and active agent. Examples of the linker, which are usually bifunctional, are 2,4-dichloropyrimidine, 4,4'-diisothiocyanoato-2,2'-stilbenedisulfoninc acid, and cyanuric chloride (2,4,6-trichloro-s-triazine). The protein is a compound which can be taken up by the tumor specifically or non-specifically, and is not recognized by the defense system of an organism as exogenous, such as autologous serum albumin. The conjugates are said to be suitable for providing a very sensitive method in nuclear medicine for the diagnosis of tumors and also offering methods for diagnosing tumors in X-ray diagnosis, computerized tomography, nuclear spin tomography, electron spin resonance spectroscopy, or electron microscopy.
U.S. Pat. No. 5,401,493 (Lohrmann et al.) discloses organic compounds for diagnostic imaging which contain at least one aryl group which has been derivatized to contain at least one perfluoro-1H,1H-neopentyl moiety. The perfluoro-1H,1H-neopentyl groups produce a single magnetic resonance to provide a maximum signal to noise ratio. A preferred perfluoro-1H,1H-neopentyl group is 3,5-(CF.sub.3).sub.3 C(CH.sub.2)-C.sub.6 H.sub.3 -. A lipid emulsion may also be provided as a carrier vehicle to deliver the derivatized analog to a mammalian recipient.
U.S. Pat. No. 5,422,094 (Antich et al. '094) discloses an .sup.19 F labelled NMR composition said to be useful in methods of NMR imaging and spectroscopy comprising a .sup.19 F-containing sensor moiety and an antibody and optionally a spacer moiety to separate the sensor moiety and the antibody. The sensor moiety comprises --COCF.sub.3 or --NHCOCF.sub.3 and produces a single .sup.19 F NMR signal. The antibody reacts specifically with a particular antigen and is bound to the .sup.19 F-containing sensor moiety. Antich et al.'094 states that the spacer moiety can be used to isolate the .sup.19 F atoms from the substrate thereby enhancing the NMR signal produced. Antich et al.'094 states that the spacer moiety can be, for example, an alkyl hydrocarbon having a chain length of approximately 1-100 carbon atoms and containing an amino group, or alternatively, the spacer moiety can be selected from the group consisting of alkyl, alkoxy, aryl, and alkaryl hydrocarbons which contain an amino group, hydrazine, hydrazide, semicarbazide, and hydroxylamine. Antich et al.'094 state that the spacer moiety can optionally include one or more .sup.19 F atoms.